This study has identified two neural pathways that play a major role in the expression of the isolation call, a stereotyped vocalization elicited by brief separation from conspecifics. One pathway, encompassing the caudal thalamic tegmentum and adjacent core gray matter, is involved with the normal structural patterning of this call. This pathway plays no significant role in the motivation to produce isolation calls or in the normal structural patterning of other vocalizations. A second pathway involves the rostral limbic cortex (cingulate and subcallosal gyrus) and adjacent frontal neocortex. This neural tissue is related to the tendency to produce isolation calls. Bilateral lesions result in a failure to vocalize when visually and acoustically isolated from conspecifics, although some tendency to vocally respond upon hearing squirrel monkey sounds is retained. Other studies have shown this same cortical area to be rich in opiate receptors. Therefore, our finding that systemic administration of an exogenous opiate, morphine, blocks the tendency to produce isolation calls suggests a role for endogenous opiates acting on neurons within this rostral midline cortex in regulating production of this vocalization. Parts of this same brain region also influence the tendency to express vocalizations associated with the mirror-directed genital display, while leaving nonvocal display components relatively unaffected. Vocalizations by adult males in this context often closely resemble infantile versions of the isolation call in their combined noisy and tonal structure and variably placed frequency modulation.